Microbubble complexes and methods of use

ABSTRACT

The present invention relates to a microbubble complex comprising a microbubble having an outer shell comprising a mixture of native and denatured albumin encapsulating a perfluorocarbon gas, a therapeutic agent, a bifunctional linker having one end attached to the therapeutic agent and the other attached to a ligand and wherein the ligand is bound to the other shell of the microbubble through hydrophobic interactions. Also included are methods for delivering the aforementioned microbubble complex to a tissue target.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/219,419, filed Jul. 26, 2016, now co-pending, which is a continuationof U.S. patent application Ser. No. 13/528,399, filed Jun. 20, 2012,which is a continuation-in-part of U.S. patent application Ser. No.13/235,890, filed Sep. 19, 2011, the entire disclosures of which areincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 28, 2012, isnamed 250944-2.txt and is 833 bytes in size.

BACKGROUND

The invention relates generally to novel binding of therapeutic agentsto albumin microbubble pharmaceuticals using an attachment of albuminaffinity ligands to the agents. The binding provides a method ofmicrobubble-assisted delivery of therapeutic agents to targeted cells ortissue of interest, either in vitro or in vivo.

Ultrasound-mediated destruction of microbubbles carrying drugs has beenfound to be useful as a noninvasive drug delivery system. Drugs or othertherapeutic agents can be incorporated into the microbubbles in a numberof different ways, including binding of the drug to the microbubbleshell and attachment of ligands. For example, perfluorocarbon-filledmicrobubbles are sufficiently stable for circulating in the vasculatureas blood pool agents; they act as carriers of these agents until thesite of interest is reached. Ultrasound applied over the skin surfacecan then be used to burst the microbubbles at this site, causinglocalized release of the drug or therapeutic agents on site specificlocations.

More specifically, albumin microbubbles have been used and delivered toa specific organ target by site-directed acoustic ultrasound. Albumin isa major protein in blood, and its natural physiological role is to bindand carry a wide variety of lipophilic/poorly soluble ligands throughoutthe body. These ligands, which may have an affinity to albumin, includefatty acids and other biosynthetic and catabolic products that arehydrophobic in nature. As such albumin microbubbles have been used tocarry a variety of therapeutic agents based on proteins and otherbiologics including, oligonucleotides (ODN) and polynucleotides such asantisense ODN, with sequences complementary to a specific targetedmessenger RNA (mRNA) sequence. These microbubble-nucleic acid complexesmay be generated from unmodified ODN that are mixed with albumin orlipid components during microbubble shell formation or alternatively,the complex formation can be performed by mixing preformed microbubbleswith an ODN of interest. In both cases, the ODN acts as a mechanisticintervention in the processes of gene translation or an earlierprocessing event. The advantage of this approach is the potential forgene-specific actions which should be reflected in a relatively low doseand minimal non-targeted side effects.

However, a key barrier to translating the potent biology of ODN intodrugs is known to be at the level of drug delivery with efficacy andsafety. For example, ODN delivery with chemical formulation, viralvectors, and particle delivery have been hampered with clinical safetyrelated problems before therapeutic efficacy can be attained.Furthermore, the use of albumin microbubbles as a carrier of ODNs suchas siRNA is limited due to the limited binding of the ODNs to thealbumin microbubble as well as the stability of the albumin-ODN complex.Due to negative shell surface potential of albumin, the negativelycharged shorter nucleic acids do not bind very well to the microbubbleand gene transfection efficiencies using these complexes are generallysuboptimal.

Thus there is a need to improve the binding of the therapeutic agents tothe microbubble as well as improving the stability and efficacy of themicrobubble complex.

Furthermore there is a need to reduce toxicity in the selective deliveryof highly cytotoxic drugs. Non-targeted delivery of these drugs cancause systemic toxicity and has prevented the use of many of these drugsall together or at higher doses required for good efficacy. Attempts todeliver these as pro-drugs in many cases have reduced this problem,however, selective uptake in the targeted tissue is not always easy toachieve as most of the uptake mechanisms in the diseased tissue are alsopresent in the normal tissue. Enhancing the uptake of these drugs inselective tissues by non-natural mechanisms as disclosed herein,therefore can add considerable value.

BRIEF DESCRIPTION

Provided herein are novel compositions and methods for increased bindingof therapeutic drugs to microbubbles using the affinity ofligand-therapeutic compositions towards the albumin shell.

Systemic circulation of the microbubbles carrying the therapeuticcomposition can be easily visualized through ultrasound imaging.Therapeutic agent is released from the microbubbles using a trigger ofhigh energy pulsed ultrasound specific to the site of treatment. Thecavitation of microbubbles causes sonoporation of the neighboringcells/tissue.

In one embodiment a microbubble complex is disclosed comprising amicrobubble having an outer shell comprising a mixture of native anddenatured albumin and a hollow core encapsulating a perfluorocarbon gas,a therapeutic agent selected from the group comprising a small moleculechemotherapeutic agent, a peptide, a carbohydrate, or anoligonucleotide, and a bifunctional linker having one end attached tothe therapeutic agent and the other attached to the ligand throughreaction of a reactive group on the ligand. The ligand is bound to theouter shell of the microbubble through hydrophobic interactions.

In another embodiment a method is for delivering the aforementionedmicrobubble complex to a tissue target is disclosed. The methodcomprising the steps of providing the microbubble complex,administrating the microbubble complex to a subject wherein the subjectis the source of the tissue target, and administering ultrasonic energyto the subject, wherein the ultrasound energy is sufficient to causecavitation of the microbubble complex in the tissue target.

BRIEF DESCRIPTION OF THE FIGURES

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying figures wherein:

FIG. 1 is a representation of non-covalent binding of siRNA-ligand toalbumin microbubbles.

FIG. 2 is representative micrograph of a gel shift assay for a mixtureof cholesterol conjugated siRNA (2 pmoles) and varying amounts ofOptison (0, 9, 22 and 46 pmoles for i, ii, iii and iv respectively).

FIG. 3 is a. representative micrograph of a gel shift assay forCy3-siRNA (4 pmoles) mixed with varying concentrations of either Optisonor native HSA which shows no shift in gel assay.

FIG. 4 is a. representative micrograph of a gel assay forCy3-cholesterol-siRNA (2 pmoles) mixed with varying concentrations ofeither Optison, native HSA or denatured HSA.

FIGS. 5A and 5B are graphical representations of binding properties ofcholesterol-siRNA to Optison and native HSA.

FIG. 6 is a graphical representation of the uptake of siRNA by U-87tumor cells in opticell is quantified by measuring cellcy3-fluorescence.

FIG. 7 are micrographs of fluorescence images showing a comparison ofsiRNA transfection between a lipid transfection reagent (RNAifect) andOptison.

FIG. 8 is a graphical representation of the mean cell fluorescencevalues and standard error of siRNA transfection between a lipidtransfection reagent (RNAifect) and Optison.

FIG. 9 is a graphical representation of the fraction bound offluorescein-myristate to Optison and native HSA as calculated fromanisotropy values.

FIGS. 10A and 10B are the fluorescein bound to Optison (0, 8, 40 and 200pmoles for i, ii, iii and iv respectively) and visualized on the gel asdark bands for fluorescein-myristate (63 pmoles) andfluorescein-stearate (180 pmoles) respectively.

DETAILED DESCRIPTION

The following detailed description is exemplary and not intended tolimit the invention of the application and uses of the invention.Furthermore, there is no intention to be limited by any theory presentedin the preceding background of the invention or descriptions of thedrawings.

The invention relates generally to microbubble-assisted delivery of atherapeutic agent to cells or tissue of interest, either in vitro or invivo.

In certain embodiment the therapeutic agent may be comprised of a smallmolecule chemotherapeutic agent, a peptide, a carbohydrate, or anoligonucleotide, and a bifunctional linker having one end attached tothe therapeutic agent and the other attached to the ligand throughreaction of a reactive group on the ligand. In certain embodiment thetherapeutic agent may be an oligonucleotide (ODN). Oligonucleotidesrefers to nucleic acid polymers that are formed by bond cleavage oflonger nucleic acids or are synthesized using building blocks, protectedphosphoramidites of natural or chemically modified nucleosides or, to alesser extent, of non-nucleosidic compounds. The length of theoligonucleotide may vary from a short nucleic acid polymer of fifty orfewer base pairs to more than 200 base pairs. As used herein, ODN alsorefers to polynucleotides having more than 200 base pairs. Also includedare antisense ODN which refer to single strands of DNA or RNA that arecomplementary to a chosen sequence. In the case of antisense RNA,antisense RNA prevents protein translation of certain messenger RNAstrands by binding to them. Antisense DNA can be used to target aspecific, complementary (coding or non-coding) RNA. Also included aresmall interfering RNA (siRNA), sometimes known as short interfering RNAor silencing RNA, is a class of double-stranded RNA molecules, typically20-25 nucleotides in length, that play a variety of roles in biologyincluding the RNA interference (RNAi) pathway, where it interferes withthe expression of a specific gene, as an antiviral mechanism, or inshaping the chromatin structure of a genome.

In certain embodiments, the therapeutic agent may be a cytotoxin. Asused herein cytotoxin refers to a substance that has a toxic effect oncells. For example a cytotoxin may cause undergo necrosis, in which theylose membrane intearity and die as a result of cell lysis. In otherexamples a cytotoxin may be associated with antibody-dependent cellmediated cytotoxicity wherein a cell is marked by an antibody and actedupon by certain lymphocytes.

Examples of cytotoxic agents are listed in Goodman and Gilman's “ThePharmacological Basis of Therapeutics,” Tenth Edition, McGraw-Hill, NewYork, 2001. These include taxol; nitrogen mustards, such asmechlorethamine, cyclophosphamide, melphalan, uracil mustard andchlorambucil; ethylenimine derivatives, such as thiotepa; alkylsulfonates, such as busulfan; nitrosoureas, such as carmustine,lomustine, semustine and streptozocin; triazenes, such as dacarbazine;folic acid analogs, such as methotrexate; pyrimidine analogs, such asfluorouracil, cytarabine and azaribine; purine analogs, such asmercaptopurine and thioguanine; vinca alkaloids, such as vinblastine andvincristine; antibiotics, such as dactinomycin, daunorubicin,doxorubicin, bleomycin, mithramycin and mitomycin; enzymes, such asL-asparaginase; platinum coordination complexes, such as cisplatin;substituted urea, such as hydroxyurea; methyl hydrazine derivatives,such as procarbazine; adrenocortical suppressants, such as mitotane;hormones and antagonists, such as alrenocortisteroids (prednisone),progestins (hydroxyprogesterone caproate, medroprogesterone acetate andmeaestrol acetate), estrogens (diethylstilbestrol and ethinylestradiol), antiestrogens (tamoxifen), and androgens (testosteronepropionate and fluoxymesterone).

Drugs that interfere with intracellular protein synthesis, proteinsynthesis inhibitors, can also be coupled to the ligand; such drugs areknown to these skilled in the art and include puromycin, cycloheximide,and ribonuclease.

In one embodiment, the protein includes, but is not limited to, enzymes,soluble and serum proteins, proteins expressed on a surface of a cell,non-immunoglobulin proteins, intracellular proteins, and segment ofproteins that are or can be made water-soluble, either individually orin combinations thereof as well as any derivatives of the proteins.

In a particular embodiment, the protein includes such as, but notlimited to, cysteine proteases, glutathione S transferase, epoxidehydrolase (EH), thiolase, NAD/NADP-dependent oxidoreductase, enoyl coAhydratase, aldehyde dehydrogenase, hydroxypyruvate reductase, tissuetransglutaminase (tTG), formiminotransferase cyclodeaminase (FTCD),aminolevulinate-dehydratase (ADD), creatin kinase, carboxylesterase(LCE), monoacylglycerol (MAG) lipase, metalloproteases (MP),phosphotases (protein tyrosine phosphotases, PTP), proteosome, FK506binding protein (FKBP12), mammalian target of Rapamycin (mTOR;alternatively known as FKBP-rapamycin binding domain (FRB)), serinehydrolase (superfamily), ubiquitin-binding protein, -galactosidase,nucleotide binding enzymes, protein kinases, GTP-binding proteins,cutinase, adenylosuccinate synthase, adenylosuccinate lyase, glutamatedehydrogenase, dihydrofolate reductase, fatty acid synthase, aspartatetranscarbamylase, acetylcholinesterase, HMG cholate reductase, andcyclo-oxygenase (COX-1 and COX-2), either individually or incombinations thereof. Also included are any derivatives of any of theproteins.

In another example, the protein is substantially free of a cofactor.“Substantially free of a cofactor” includes proteins that do not requireany additional cofactor, chemical, chemical modification, or physicalmodification to be naturally stable under physiological conditions androom temperature and pressure in solution or as a solid, and can bindits corresponding ligand in vivo.

In one embodiment, an albumin microbubble may be utilized to carry atherapeutic agent in systemic delivery. Tissue targeted ultrasoundacoustic energy may then be used to cavitate the albumin microbubble anddeliver the therapeutic agent into the intracellular environment. Forexample the microbubble complex may be administered intravenously orinto the peritoneum (intraperitoneally) of a subject whose cells ortissues are to be targeted. Once the microbubble complex is carriedthrough the subject to the targeted cell, the ultrasound acoustic energyis delivered. In certain embodiments, visualization of the targetedcells may occur prior to delivering the ultrasound while in still otherembodiments visualization may be performed in real time and thecavitation monitored.

In certain embodiments, the albumin outer shell of the microbubble iscomprised of both native and denatured albumin held together by mostlycysteine to cysteine bonds. In certain embodiments, the primarycomposition of the albumin shell is mostly in the native form whereinthe denatured portion allows for increased cysteine bond attachments. Incertain embodiments the relative amount of denatured albumin to nativealbumin ranges from approximately 0.5 to 30 wt %. In other embodimentsthe relative amount is in the range of approximately 1% to 15 wt %. Themixture of native and denatured albumin provides a balance of shellelasticity needed for cavitation, with increased reactive binding siteson the microbubble surface. The microbubbles are formed by sonication ofperfluorocarbon gas in the presence of pre-heated albumin solution. Asmall part of the albumin molecules rearrange during sonication ofpre-heated albumin solution and crosslinking occurs through disulfidelinkages between albumin molecules. These albumin molecules are believedto be similar in structure to an F form of albumin which has morehydrophobic residues exposed. This allows increased binding sites forhydrophobic interactions.

In certain embodiments, the microbubble may be filled with an insolubleperfluorocarbon gas, such as but not limited to, perfluoromethane,perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, ora combination thereof. In certain embodiments, the microbubbles areabout 1 to about 5 microns in diameter, the size being optimized toallow circulation through the blood stream.

In certain embodiments, the therapeutic-microbubble complexes comprise atherapeutic agent modified with a linker having a reactive group capableof binding with a ligand having affinity towards albumin. As such, thetherapeutic agent may be coupled to albumin though the ligand.

The linker includes any linking moiety that attaches the ligand to thetherapeutic agent through a first moiety. The linker can be as short asone carbon or a long polymeric species such as polyethylene glycol,tetraethylene glycol (TEG), polylysine or other polymeric speciesnormally used in the pharmaceutical industry for modulatingpharmacokinetic and biodistribution characteristics of therapeuticagents. Other linkers of varying length include C1-C250 length with oneor more heteroatoms selected from O, S, N, P, and optionally substitutedwith halogen atoms. In a particular embodiment, the linker comprises atleast one of an oligomeric or polymeric species made of natural orsynthetic monomers, oligomeric or polymeric moiety selected from apharmacologically acceptable oligomer or polymer composition, an oligo-or poly-amino acid, peptide, saccharide, a nucleotide, and an organicmoiety with 1-250 carbon atoms, either individually or in combinationthereof. The organic moiety with 1-250 carbon may contain one or moreheteroatoms such as O, S, N or P and optionally substituted with halogenatoms at one or more places.

The first moiety may simply be an extension of the linker, formed by thereaction of a reactive species on the linker with a reactive group onthe therapeutic agent. Examples of reactive species and the reactivegroup include, but are not limited to, activated esters (such asN-hydroxysuccinimide ester, pentafluorophenyl ester), a phosphoramidite,an isocyanate, an isothiocyanate, an aldehyde, an acid chloride, asulfonyl chloride, a maleimide an alkyl halide, an amine, a phosphine, aphosphate, an alcohol or a thiol with the proviso that the reactivespecies and reactive group are matched to undergo a reaction yieldingcovalently linked conjugates.

In certain embodiments, the reactive moiety may be a primary aminefunctionality, and as such, the amine modified therapeutic agent may beconjugated to the affinity ligand through reaction of a carboxyl moietyof the ligand. In certain other embodiments, the reactive moiety may bean alcohol attached to the ligand through a phosphate group.

The affinity ligand includes fatty acids, steroids, small aromaticcompounds or a combination thereof. Examples of albumin bindingmolecules may be found in US patent application publication number2010/0172844, published Jul. 8, 2010.

For example in certain embodiments the affinity ligand is a fatty acid,including but not limited to myristoyl, lithocolic-oleyl, docosanyl,lauroyl, steoroyl, palmitoyl, oleoyl, or linoleoyl. In otherembodiments, the lipophilic molecule is a steroid or modified steroidincluding, cholesterol, cholic acid, lithocholic acid, orchenodeoxycholic acid. In other embodiment, the high affinity moleculeis selected from 4-p-iodophenyl-butyric acid and analogs or derivativesthereof. In still other embodiments, the therapeutic agent comprisessiRNA, the linker comprises tetraethylene glycol, and the ligandcomprises cholesterol.

In certain embodiments, the therapeutic-albumin complexes may beprepared either by sonicating ligand-modified therapeutic agent withalbumin or lipid in the presence of perfluorocarbon or by mixingpreformed bubbles with ligand modified therapeutic agents. In certainembodiments, these molecules may be attached to therapeutic agents ofinterest during therapeutic agent synthesis. For example thephosphoramidites of cholesterol may be used to incorporate cholesterolduring DNA or RNA synthesis on a nucleic acid synthesizer, or postsynthesis by incorporating a reacting moiety.

In certain embodiments, the therapeutic agent is a modified ODN whichmay be prepared enzymatically by using modified nucleosidetriphosphates; modified either with the ligand itself or with a reactivefunctionality for post synthesis modification with the ligand. Ligandattachment may be at one or both termini, internal to the nucleic acidsequence or at multiple positions depending upon the ODN use. In certainembodiments, where siRNA is the ODN, attachment may be through the 3′ OHposition.

In certain embodiments, in addition to the ligand, the therapeuticagent, including where the therapeutic agent is ODN, may be selectivelymodified to protect from nucleases. In certain embodiments, stabilizingmodification may include phosphorothioate modification, or 2′-OMemodification.

In certain embodiments, the microbubble complexes may be incubated withcells or the tissue of interest or injected into the body, preferablyintravenously, and then cavitated with ultrasound energy at desired siteand at a predetermined time or during live imaging.

In certain embodiments, the microbubble complex may be viewed duringsystemic travel in the blood circulation under normal ultrasounddiagnostic imaging. When the bubble arrives on tissue target, in thiscase the tumor, a series of pulsed acoustic energy waves are sent to thetumor. This creates inertial cavitation on the microbubble, whichcollapses the microbubble. Cavitation of the microbubble occurs wherethe acoustic energy is maximally located. This direction is achieved onthe ultrasound probe by parameters related to mechanical index force,optimal ultrasound acoustic distance, and dimensions of the ultrasoundacoustic sweep. The force generated can then potentially form microporeswithin the cellular plasma membrane. Typically the pulsed enemy isadministered at a frequency of about 0.5 to about 5 MHz.

These micropores, along with the microjetting force created underinertial cavitation, may facilitate the entrance of ODN into thecellular cytoplasmic environment. For example when the ODN is siRNA,siRNA in the intracellular environment will utilize the host machineryto silence mRNA and later protein synthesis. Similarly, where mRNAmessage acts as an angiogenesis promoting proteins including vascularendothelial growth factor (VEGF), the reduction of VEGF expression in atumor may halt or slow tumor growth. After microbubble cavitation thedense gas of the microbubble center is exhaled out of the body and thealbumin shell is metabolized and excreted via the liver eliminationpathway.

In an exemplary embodiment, a bolus of the microbubble complex may bemixed to an optimal ratio from previous therapeutic investigations. Oncethe mixture of the complex is established, the bolus drug is injectedsystemically by venous route.

For example in the use of a siRNA-microbubble bolus, the bolus may bemonitored in the first pass blood kinetics. The microbubble resonanceand thus enhanced ultrasound contrast may be monitored with anultrasound probe using low diagnostic levels of acoustic energy. Duringcirculation the bolus arrives on organ target. Cardiovascular tissueperfusion may assist in delivering the bolus into deep microvessels withsmall lumen diameters. By supplying pulsed acoustic energy, sufficientenergy may be provided for the microbubble to undergo inertialcavitation. Once the microbubble cavitation is complete the siRNAcontents may be delivered across the plasma membrane and into thediseased cell. While in the cytoplasmic intracellular environment thesiRNA can have a therapeutic effect.

During microbubble cavitation siRNA entrance into the cell may occur byvarious mechanisms. For example the siRNA may enter the cell by a: amicrojetting force from the collapsing microbubble which can push siRNAinto the cytoplasm. Alternatively the mechanism may include microjettingenergy or sonoluminescence energy which creates temporary microporeswithin the plasma membrane to allow for passive diffusion of the siRNAinto the cell, or during microbubble resonance before actual cavitationthe microbubble bumping into the plasma membrane may push the siRNA inthe cell.

As such, the mechanism of microbubble delivery has potentialapplications in the treatment of a wide variety of diseases, which caninclude cancer, inflammatory, infectious, cardiovascular, metabolic,autoimmune, and central nervous system diseases. Many of these diseasescannot currently be effectively treated by virtue of targeting molecularmechanisms not accessible to conventional small molecule drugs andmonoclonal antibodies.

Experimental

EXAMPLE 1

FIG. 1 is a representation illustration of binding of siRNA to albuminencapsulated microbubbles to form a microbubble-siRNA complex.

The target siRNA for VEGF silencing (vascular endothelial growth factor)were synthesized by IDTDNA technologies. IDTDNA provided lipidmodifications such as cholesteryl TEG on the siRNA (Chol-siRNA) as wellas dye conjugation.

Sense strand: (SEQ ID NO: 1) 5′-Cy3/GCAUUUGUUUGUCCAAGAUmUmU/3′-LipidAntisense strand; (SEQ ID NO: 2)5′/mAmArA rUrCrU rUrGrG rArCrA rArArC rArArA rUrGrC/3′

Cyanine dye, Cy3 on the siRNA has an excitation wavelength of 550 nm anda peak emission of 580 nm. The siRNA has been labeled with a cy3 dye foreasy visualization of siRNA during binding assays and othercharacterization techniques. Optison™ (GE Healthcare, Chalfont St.Giles, United Kingdom, 10 mg/ml albumin) was centrifuged; the top layerwas discarded and the excess albumin solution in the bottom was used forthe binding studies. Lyophilized human serum albumin (HSA) powder (SigmaAldrich, St. Louis Mo.) was dissolved in 1× phosphate buffered saline(PBS) to make a stock solution of 10 mg/mL. Both Optison and nativealbumin solution dilutions were prepared with 1× PBS. Denatured HSAsolution was prepared by heating native ESA solution to 80° C. for 20minutes.

Binding Reaction:

The stock solutions of cy3-siRNA and cy3-siRNA-cholesterol, 20 μM wereprepared in RNAse free water and stored at −20° C. 4 pmoles of cy3-siRNAand 2 pmoles of cholesterol-siRNA solutions were mixed with varyingamounts of Optison solution, native HSA and denatured HSA solution,ranging from 0 to 50 pmoles. The reaction buffer was 1× PBS, pH 7.4. Thereaction mixture was incubated under dark at 25° C. for 45 minutes.After incubation, ten μL of siRNA mixtures was mixed with 2 μL of Novex®Hi-Density TBE Sample Buffer (5×) (Invitrogen, Carlsbad, Calif., USA).

Gel Electrophoresis:

All the reactions were loaded onto precast 6% nondenaturingpolyacrylamide gels (Invitrogen, Carlsbad, Calif., USA). The gel was runat 100 V for 45 min in 0.5× Novex TBE Running buffer (Invitrogen,Carlsbad, Calif., USA). The gels containing either DNA, protein or bothwere imaged for cy3 fluorescence using a typhoon scanner (Typhoon™9410,GE Healthcare).

Results:

The fluorescence of Cy3 attached to siRNA can be visualized as distinctsiRNA bands on the gel. When a mixture of siRNA and albumin solution wasrun on the gel, the mobility of albumin bound-siRNA is slower thanfree-siRNA resulting in two bands on the gel. In preliminary trials,sypro ruby stain from EMSA kit (Molecular Probes, Eugene, Oreg., USA)was used to observe the albumin bands in the gel. An example is shown inFIG. 2 which is a gel shift assay for a mixture of cholesterolconjugated siRNA (2 pmoles) and varying amounts of Optison (0, 9, 22 and46 pmoles for i, ii, iii and iv respectively). Fluorescence imaging ofthe gel shows distinct bands for siRNA (lower band sections) and albumin(upper band sections).

Cy3-siRNA

When the mixture of cy3-siRNA and native HSA/Optison was run on the gel,there was no bound-siRNA visualized for increasing concentrations ofalbumin. There was no significant binding of cy3-siRNA with eithernative HSA or Optison solution. This is shown in FIG. 3 which is afluorescence image of a gel for Cy3-siRNA (4 pmoles) mixed with varyingconcentrations of either Optison or native HSA shows no shift in gelassay. The dark bands on the gel are the cy3-fluorescence on siRNA.There is no significant binding of cy3-siRNA to both Optison and nativeHSA.

Chol-siRNA

FIG. 4 shows the gel images for binding of chol-siRNA with Optison,native HSA and denatured HSA. Chol bound to both native HSA and Optisonsolution, while the binding significantly decreased for the same amountof denatured HSA. The fluorescence intensity of siRNA in each lane wasestimated manually by drawing a box around the bands. Background,equivalent to the average intensity value of the gel, was subtractedfrom the intensity value of each siRNA band. Fluorescence intensities ofbound siRNA over a wide range of albumin concentrations were calculated.Relative fluorescence, R was calculated as:R=(Fbound-Ffree)/Ffree  (1)

Fbound is fluorescence intensity of bound-siRNA band and Ffree isfluorescence intensity of free-siRNA band. Relative fluorescence wasplotted albumin concentration. This is shown in FIGS. 5A and 5B, whichare graphical comparisons of binding properties of cholesterol-siRNA toOptison and native HSA as described below.

At low albumin concentration ranging from 0 to 15 μM, linear dependenceof bound fluorescence on albumin concentration was visualized. FIG. 5Ashows that at this concentration range, the amount of chol-siRNA boundto Optison solution was higher than the binding to native HSA. Toestimate binding constants, higher concentration of albumin was used toallow saturation of amount of siRNA bound to albumin. Fraction bound, xis determined as:x=(Fbound−Ffree)/(Fsat−Ffree)  (2)Fsat is the fluorescence intensity of maximum bound-siRNA undersaturation conditions.

Fraction bound was plotted against increasing albumin concentrations, asshown in FIG. 5B, and the data points were fitted to the followingequilibrium equation;x=n*[Albumin],/(kd±[Albumin])  (3)kd is the dissociation constant, n is the number of binding sites and[Albumin] is the total albumin concentration for the respective samples.Equation 3 was solved using a non-linear fit to determine kd and n forbinding of chol-siRNA to both Optison and native HSA (Table 1).Microsoft Excel's solver tool was utilized for this non-linear fit, andthe sum of squared errors (SSE) was found to be 0.07 and 0.06 forOptison and native HSA respectively. The binding constant of chol-siRNAwas similar for both Optison and native HSA.

EXAMPLE 2

Delivery of siRNA to Tumor Cells

Cell Culture:

MATBIII rat mammary carcinoma and U-87 human glioblastoma cells werecultured in McCoy's 5A Medium (modified) (1×) (Invitrogen, Carlsbad,Calif., USA) and Eagle's Minimum Essential Medium (EMEM) (ATCC,Manassas, Va.) respectively. Both the media solutions were supplementedwith 10% heat deactivated fetal bovine serum (FBS) (Fisher Scientific,Springfield, N.J.) and 1% penicillin-streptomycin (Sigma Aldrich, StLouis, Mo.). The cells were maintained at 37° C. in a humidifiedatmosphere with 5% CO2.

Sonication of Substrate-Attached Cells:

MATB-III and U-87 cells were grown in 10 mL capacity Opticell units(Nalge Nunc International, Rochester, N.Y.) to 90% confluence. The mediain OptiCell was replaced with 10 mL fresh media containing 40 pmoles ofeither cy3-siRNA or cholesterol-siRNA. The opticell was left in theincubator for 24 hours at 37° C. Separately, the cells were eithertreated with siRNA solution mixed with Optison microbubbles (300 μL) ora lipid transfection reagent (90 μL) (RNAifect, Qiagen, Valencia,Calif.). For siRNA/Optison mixtures, Vivid i imagers with a cardiacsector probe (3S) was used to rupture the microbubbles and deliver thesiRNA drug from microbubbles. The opticell was immobilized in a waterbath, and the ultrasound probe was attached to a motion arm that spannedthe entire length of the opticell. The tip of the probe was immersed inwater, and the distance between the probe and opticell surface was 3 cmthat allowed sonication of the entire width of the opticell. Themicrobubbles in opticell were treated with a mechanical index, MI>1.3continuous sonication. The probe moved at a speed of 1 m/s over theentire length of the opticell. After sonication, the cells wereincubated for 24 hours at 37° C. Similarly, the cells treated withRNAifect were also kept in the incubator for 24 hours. After incubation,the cells were imaged using a fluorescence microscope (Zeiss AxioImager.Z1, Carl Zeiss). The filter used for cy3 was DsRed/Cy3 (546ex/620 em). In the region of interest (ROI) in the fluorescent images,cell fluorescence was measured and the mean values of cell fluorescencewere calculated. ImageJ was used to process the images and calculatefluorescence intensities.

The data are reported as mean+1.0 standard error (SE) for N=4. Thestatistical significance of the differences between the groups wasevaluated using two-sample t-test and the statistical analyses werecarried out using Minitab® 12 (Minitab Inc, State College, Pa. USA).

Results:

The effect of the delivery system is illustrated in FIG. 6 for U-87cells incubated with either cy3-siRNA or chol-siRNA. The delivery ofsiRNA into the tumor cells is represented by average cellcy3-fluorescence. For each group, mean fluorescence values and standarderrors are reported in. Cell sonication substantially enhanced cy3-siRNApenetration into the cell. Due to the effects of sonoporation, averagecell fluorescence for Optison/ultrasound treated cells was 39% more thanuntreated cells. For cholesterol-siRNA, there was a 53% increase inaverage cell fluorescence after treatment with Optison/ultrasound.Significant differences between the groups were evaluated using twosample t-tests (p=0.032 for cy3-siRNA and p=0.059 forcholesterol-siRNA).

Similarly for MATBIII cells, the effect of Optison/ultrasound treatmentwas compared to a commercially available lipid transfection reagent. Thecells were treated with either Cy3-siRNA or chol-siRNA in combinationwith either RNAifect or Optison/ultrasound delivery agents and theresults are shown in FIGS. 7 and 8. FIG. 7 shows representative imagesof the cells after treatment. FIG. 8 reports the mean cell fluorescencefor all groups with standard errors represented as error bars. Forcy3-siRNA, the average cell fluorescence was higher forOptison/ultrasound treatment (two sample t-test, p=0.007). This isprimarily due to sonoporation of the cells in the presence ofmicrobubbles. There was no significant difference between RNAifect andOptison/ultrasound delivery of chol-siRNA into cells. Although theaverage cell fluorescence was similar, the lipid transfection reagentwas found to be toxic to the tumor cells as evidenced by the irregularshape of the cells in FIG. 7. It should be noted that the same amount oftransfection reagent and siRNA was used in both unmodified andcholesterol-siRNA. While the transfection reagant was toxic in both thecases, it was higher for chol-siRNA.

EXAMPLE 3

Preliminary binding studies of therapeutic-fatty acid conjugates tomicrobubbles were evaluated using fluorescein—fatty acid conjugates.

Conjugation Method;

Fatty acid NHS ester (2 equivalents, 5.37 mg Myristic acid NHS ester or6.38 mg Stearic acid NHS ester was taken in a 50:50 mixture of DMSO anddichloromethane (100 ul) and mixed with a solution of Fluoresceincadaverine (FL-Cadaverine, 5 mg, 1 equivalent, in 50 ul DMSO). To thisdiisopropylethyl amine (3.8 equivalents) was added and mixture wasvortexed to give a clear solution. Samples were kept in the dark at roomtemperature. After 4.5 h, reaction was checked by HPLC and was found tobe complete. A large shift in retention time was observed for bothconjugates (Retention times FL-Cadaverine 4.7 min, FL-Cadaverinestearate 12.1 minute and FL-Cadaverine Myristate 9.9 min, columnX-Bridge Shield RP 18, 4.6×50 mm column, particle size 5 um, gradientmethod 0-100% B in 15 min and 100% B for 5 min, solvent A 0.1M TEAA, pH7.0 and solvent B 100% acetonitrile, flow rate 1 mllmin) as expected.Crude product was diluted with DMSO to ˜2 ml and purified on AKTApurifier using Xterra MS C18, 19×100 mm column and a gradient of 0-100%B in 18.75 column volumes at a flow rate of 10 ml/min. Solvent A and Bwere as described above for the analytical method. Product was collectedin multiple fractions and each fraction was analyzed by analytical HPLC.Only the purest fraction in each case (˜90% purity) was used for bindingstudies (starting material itself was ˜86% pure, remaining likely aregioisomer with same spectral properties). This fraction wasconcentrated to dryness at room temperature. Residue was suspended inwater (˜2 ml) and extracted with dichloromethane (3×2 ml). Organicextracts were combined, dried over anhydrous sodium sulfate andconcentrated to dryness.

Fluorescence Polarization Assay

The stock solution of fluorescein-myristate was prepared in 1× PBS. Theconcentration of fluorescein was kept low for the fluorescencepolarization assay, at 126 nM. Varying concentrations of either Optisonor HSA solutions, ranging from 0 to 15 μM albumin concentrations, wereadded to the fluorescein myristate solution. The reaction buffer was 1×PBS, pH 7.4. The reaction mixture was incubated under dark at 25° C. for15 minutes. After incubation, the changes in raw anisotropy values offluorescein were measured using a microplate reader (SpectraMax 5,Molecular Devices, Sunnyvale, Calif.).

The samples were measured in Corning 96-well plates (black plate with aclear bottom) (Sigma Aldrich, St Louis, Mo.). Fluorescein was excited at470 nm, and emission was measured at 540 nm. Fraction bound (x) wascalculated using the same equation as before (Equation 2), but replacingfluorescence values with anisotropy values. The fraction boundcalculated was then plotted against albumin concentration as shown inFIG. 9. The data are reported as mean+1.0 standard error (SE) for N=3.Equation 3 was used to determine kd and n for fluorescein-myristatebinding to both Optison and native HSA (Table 2). This is representedalso in FIGS. 10A and 10B which show the fluorescein bound to Optison(0, 8, 40 and 200 pmoles for i, ii, iii and iv respectively) isvisualized on the gel as dark bands for fluorescein-myristate (FIG. 10A)(63 pmoles) and fluorescein-stearate (FIG. 10B) (180 pmoles).

When the fluorescein without the myristate conjugation was tested forits binding properties to albumin, no significant changes in anisotropywas observed. It is well known that the fatty acids have strongerbinding properties than cholesterol, and is also confirmed here with thelower dissociation constants, kd, observed for fluorescein-myristateconjugate (Table 1 and Table 2).

TABLE 1 Number of binding sites and dissociation constants for bindingof cholesterol-siRNA to Optison and native HSA Optison Native HSA Numberof binding sites, n 1.16 1.13 Dissociation constant, k_(d) (μM) 7.16 6.4

TABLE 2 Number of binding sites and dissociation constants for bindingof fluorescein-myristate to Optison and native HSA Optison Native HSANumber of binding sites, n 1.03 1.02 Dissociation constant, k_(d) (μM)0.238 0.378

Therefore, conjugating a fatty acid such as myristate to a therapeuticcompound can increase the binding of such therapeutic compounds to thealbumin shell microbubbles. The dissociation constant offluorescein-myristate binding to Optison was lower than that of bindingto native HSA. This suggests better hydrophobic binding properties ofmicrobubble shell that has both native and partially denatured albumin.

EXAMPLE 4

Stability of siRNA In Vivo:

The stability of therapeutic compounds such as siRNA is very low onceinjected into the body. A comparison between subcutaneous and tail-veininjection of a mixture of albumin microbubbles and native siRNA (withoutconjugates) was studied.

Eleven to fourteen weeks (body weight ˜30 g) old nu/nu mice wereobtained from Charles River Laboratories (Wilmington, Mass.). Animalswere housed in accordance with the Guide for the Care and Use ofLaboratory Animals as adopted by the National Institutes of Health.Lewis lung carcinoma cells (LLC) were inoculated subcutaneously to theright flank of anaesthetized mice (3.5×106 cells/100 μl/mouse).

On the fourth day after inoculation, the mice were treated withanti-VEGF siRNA (Sigma Life Sciences, St. Louis, Mo.)—microbubblemixtures, a siRNA dose of 1.0 mg/kg for subcutaneous injections and adose of 2.0 mg/kg for tail-vein injections. The injection mixturecontained 100 μL of microbubble solution and 100 μL of siRNA in RNAsefree water. After injection, the tumors were sonicated using Vivid iimagers with a cardiac sector probe (3S). The energies were delivered ina pulsatile form with the peak MI at 1.3. Control group did not receiveany treatments.

After 24 hours from the treatment day, the mice were euthanized and thetumors were extracted. The tumors were frozen immediately and stored at−20° C. The tumors were thawed out at room temperature on the day ofVEGF measurement. The tumors were then lysed in RIPA buffer (proteaseinhibitors added) using a lysing matrix tube (Lysing matrix tube A, RPBiomedical). The lysate collected from the samples were then diluted,and measured for total protein using a protein kit (Pierce BCA reagentprotein assay kit) and for VEGF using an ELISA kit (Mouse VEGF ELISAkit, RayBiotech, Norcross, Ga.).

The results are reported in Table 3 as mean pg VEGF/mg protein forcontrol and different treatment groups. The subcutaneous injection of1.0 mg/kg—microbubble mixture resulted in an approximately 39% decreasein VEGF when compared to the control group (two-sample t-test;p=0.0096). While there was only a minor difference between the controlgroup and 2.0 mg/kg tail-vein injection of siRNA-microbubble mixtures,this may be due to lack or less efficient binding of unmodified siRNA tothe microbubble.

TABLE 3 The effect of siRNA delivery to tumors; mean pg VEGF/mg totalprotein. Mean pg VEGF/mg Conditions protein Std error 95% Cl n control296.19 24.89 48.79 5 Optison/ 181.97 24.38 47.78 7 siRNA SQ 1 mg/kgOptison/ 255.1 51.06 100.07 3 siRNA TV 2 mg/kg

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects asillustrative rather than limiting on the invention described herein. Thescope of the invention is thus indicated by the appended claims ratherthan by the foregoing description, and all changes that come within themeaning and range of equivalency of the claims are therefore intended tobe embraced therein.

The invention claimed is:
 1. A method for delivering a microbubblecomplex to a tissue target comprising the steps of: providing amicrobubble complex, said complex comprising; a microbubble having anouter shell comprising a mixture of native and denatured albumin and ahollow core encapsulating a perfluorocarbon gas; a therapeutic agentselected from a small molecule chemotherapeutic agent, peptide,carbohydrate, oligonucleotide, cytotoxin, protein synthesis inhibitor,or combination thereof; a bifunctional linker having one end attached tothe therapeutic agent and the other attached to a ligand throughreaction of a reactive group on said ligand; and wherein the ligand isbound to the outer shell of the microbubble through non-covalent,hydrophobic interactions; administrating the microbubble complex to asubject wherein the subject is the source of the tissue target; andadministering ultrasonic energy to the subject, wherein said energy issufficient to cause cavitation of the microbubble complex in the tissuetarget.
 2. The method of claim 1, wherein the tissue target is in vivoand administrating the microbubble complex comprises intravenous orintraperitoneal injection of the microbubble complex.
 3. The method ofclaim 1, further comprising the step of visualizing the microbubblecomplex at the tissue target prior to administering the ultrasonicenergy for cavitation of the microbubble complex.
 4. The method of claim1, wherein the visualizing and administering the ultrasonic energy areperformed in real time.
 5. The method of claim 4, wherein the tissuetarget is in vitro.
 6. The method of claim 1, wherein the therapeuticagent comprises siRNA, the linker comprises tetraethylene glycol, andthe ligand comprises cholesterol.